The 26S proteasome is the primary site for protein degradation in mammalian cells and is critical in cell regulation, protein quality control, and antigen presentation. Although proteasome inhibitors are widely used as research tools and in treatment of certain cancers, the mechanisms by which proteasomes catalyze the ATP- dependent degradation of ubiquitinated proteins are still poorly understood. Our recent studies defined several new ATP-dependent steps in this process. In coming years, we hope to learn how these various steps are regulated and coordinated to ensure efficient degradation of different types of proteins. After binding to the 26S, ubiquitin (Ub) conjugates activate their own degradation by stimulating ATP hydrolysis and gate opening via interactions with either of the 19S-associated deubiquitinating enzymes, Usp14 or Uch37. It will be important to understand further their regulatory roles, and how they coordinate substrate deubiquitination and proteolysis. Protein breakdown by the 26S is tightly linked to ATP hydrolysis, and we have shown that the six 19S ATPase subunits hydrolyze ATP in an ordered manner. We plan to critically test this novel cyclic mechanism and to learn if other AAA ATPases operate in a similar manner. We also recently found that prions (PrPSc) aggregates disrupt proteasome function by inhibiting gate opening and substrate entry into the 20S particle. We shall explore whether the soluble protein aggregates that appear to cause other neurodegenerative diseases cause a similar inhibition of proteasome gating. Related studies will explore how three newly discovered modifications of proteasome structure regulate 19S activity: 1) we recently found that the Ub-binding subunit Rpn13 is continually poly-ubiquitinated by 26S- associated Ub ligases, and that this modification inhibits conjugate degradation. 2) Upon excitatory stimulation of neurons, the ATPase subunit Rpt6 becomes phosphorylated, and this modification is important in synaptic plasticity. 3) The 19S caps on singly and doubly capped forms of the 26S proteasomes appear to differ in content of certain subunits. We hope to clarify the functional consequences of these intriguing proteasome modifications and learn how they influence different steps in conjugate degradation. Unlike mammalian cells, bacteria catalyze protein degradation by ubiquitin-independent, ATP-requiring protease complexes. Mycobacterium tuberculosis (Mtb) has two such ClpP genes and two regulatory AAA ATPases, ClpC1 and ClpX, which are all attractive new drug targets. We discovered that ClpP1 and ClpP2 encode a novel 2-ring complex, ClpP1P2, with a unique assembly mechanism. We now hope to solve its structure and define the detailed mechanisms of these two types of AAA ATPases and of the promising new antibiotic, Novo23, that we found uncouples ATP hydrolysis by ClpC1 from proteolysis by ClpP1P2.